Candida albicans is the leading cause of fungal infections in the US and, Candida spp. now rank fourth among all pathogens in causing nosocomial infections. Virulence in this organism is probably multi-factorial and tissue-specific, since C. albicans is capable of infecting a number of diverse environmental sites (for example, the acid vaginal canal vs. the neutral pH of blood and tissues). Two-component, histidine kinases (HK) are environmental sensor, phosphorelay proteins of prokaryotes and lower eukaryotes whose activity results in transcriptional activation of genes associated with an adaptive response to stress. Among the HK genes of pathogenic fungi, recently, we isolated a C. albicans hybrid HK (CaHK1), which is unique structurally and functionally among all HK genes thus far described. A cahk1 null strain has been constructed and compared phenotypically in vitro and in vivo to parental and gene-reconstituted strains. In vitro, hyphae of null strains flocculate at pH 7.5, indicating that cell surface changes in hyphae are associated with a mutation in caHK1 The null is also avirulent in a systemic murine model but virulent in a rat vaginal mucosal model when compared to parental and gene- reconstituted strains. While Northern analyses indicate that CaHK1 is expressed in both acid and neutral pH media, we hypothesize that activity of Cahk1p may depend upon its state of phosphorylation. There are 3 specific aims in this proposal. 1. The influence of environmental factors (temp, pH, osmolarity) on the expression of CaHK1 will be measured in vitro using Northern analyses; post-translational modifications of Cahk1p will be determined. 2. We will evaluate the virulence of CaHK1 strain constructs in oral and vaginal models in order to extend our knowledge on the role of Cahk1p in virulence. 3. Cahk1p interactions with other proteins including down-stream effector proteins are to be identified. In summary, these studies will partially define the Cahk1p phosphorelay pathway and function of CaHK1 in the pathogenesis of candidiasis.